Selectable markers are genes that impart a unique characteristic to transfected organisms which is evident during a biochemical or chemical assay. For example, some selectable markers confer (upon cells transfected with the marker) a unique resistance to cytoxic agents such as antibiotics. When a selectable marker is combined with other exogenic genes (or cDNA), the marker's conferred characteristic (i.e. antibiotic resistance) can be used to identify and select the genetically altered cells from a mixed population. This ability makes selectable markers an important tool in the study and manipulation of genes. A variety of such markers have been identified and are currently in use. (See, Wei, K., and B. E. Huber. 1996. Cytosine deaminase gene as a positive selection marker. J Biol Chem. 271(7):3812–61; Baumann, R. P., D. H. Sherman, and A. C. Sartorelli. 2002. Novel selection marker for mammalian cell transfection. Biotechniques. 32(5):1030–36; Eglitis, M. A. 1991. Positive selectable markers for use with mammalian cells in culture. Hum Gene Ther. 2(3):195–201.)
However, not all genetic markers are appropriate for all cell types or experimental systems, nor are they necessarily simple and easy to use. For example, thymidine kinase requires the use or generation of cells deficient in this enzyme. (See, Wigler, M., S. Silverstein, L. S. Lee, A. Pellicer, Y. Cheng, and R. Axel. 1977. Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells. Cell. 11(1):223–32.) Others markers may not be easily expressed in certain cell types or may require unique media. Some cell types may exhibit an innate resistance to the selecting drug.
The most commonly used DSM is the bacterial aminoglycoside phosphotransferase II gene (commonly referred to as the “neomycin-resistant” or “neo” gene), which confers resistance to the cytotoxic effect of the aminoglycoside antibiotic G418. (See, Southern, P. J., and P. Berg. 1982. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1(4):327–41.) However, the high cost for G418 makes the neo gene marker less ideal for high throughput protocols. In addition, G418 causes mammalian cells to release GPI-anchored proteins making it unsuitable for the study of these proteins. (Kung, M., B. Stadelmann, U. Brodbeck, and P. Butikofer. 1997. Addition of G418 and other aminoglycoside antibiotics to mammalian cells results in the release of GPI-anchored proteins. FEBS Lett. 409(3):333–8.) A need exists for a DSM which can be used in a variety of mammalian cells which is both easy to use and is cost effective.
The present invention identifies a novel DSM, bacterial Inosine 5′-monophosphate dehydrogenase (IMPDH), which can be used for stable transfection of mammalian cells. The advantages of the present selectable marker over existing markers include the simplicity of its selection protocol, its ability to be transfected and expressed in a variety of mammalian cell types, and the cost-effectiveness of its selection drug mycophenolic acid (MPA) relative to G418 making it suitable for high throughput screenings.
IMPDH catalyzes the oxidation of IMP to XMP (xanthosine 5′-monophosphate), which is the rate-limiting step in the de novo guanine nucleotide biosynthesis pathway, and as a result is essential for the growth and proliferation of every cell. (See, Antonino, L. C., K. Straub, and J. C. Wu. 1994. Probing the active site of human IMP dehydrogenase using halogenated purine riboside 5′-monophosphates and covalent modification reagents. Biochemistry. 33(7):1760–5; Hedstrom, L. 1999. IMP dehydrogenase: mechanism of action and inhibition. Curr Med Chem. 6(7):545–60.)
Although IMPDH is evolutionarily conserved, the bacterial enzyme is orders of magnitude more resistant than mammalian IMPDH to the toxic effect of MPA. (See, Collart, F. R., J. Osipiuk, J. Trent, G. J. Olsen, and E. Huberman. 1996a. Cloning, characterization and sequence comparison of the gene coding for IMP dehydrogenase from Pyrococcus furiosus. Gene. 174(2):209–16; Collart, F. R., J. Osipiuk, J. Trent, G. J. Olsen, and E. Huberman. 1996b. Cloning and characterization of the gene encoding IMP dehydrogenase from Arabidopsis thaliana. Gene. 174(2):217–20; Hager, P. W., F. R. Collart, E. Huberman, and B. S. Mitchell. 1995. Recombinant human inosine monophosphate dehydrogenase type I and type II proteins. Purification and characterization of inhibitor binding. Biochem Pharmacol. 49(9):1323–9; Kerr, K. M., and L. Hedstrom. 1997. The roles of conserved carboxylate residues in IMP dehydrogenase and identification of a transition state analog. Biochemistry. 36(43):13365–73; Zhou, X., M. Cahoon, P. Rosa, and L. Hedstrom. 1997. Expression, purification, and characterization of inosine 5′-monophosphate dehydrogenase from Borrelia burgdorferi. J Biol Chem. 272(35):21977–81; Zhang, R., G. Evans, F. J. Rotella, E. M. Westbrook, D. Beno, E. Huberman, A. Joachimiak, and F. R. Collart. 1999. Characteristics and crystal structure of bacterial inosine-5′-monophosphate dehydrogenase. Biochemistry. 38(15):4691–700.)
Based on this property, the inventors have demonstrated that expression of bacterial IMPDH in mammalian cells confers in them resistance to MPA toxicity and moreover that this enzyme is a useful DSM for the expression and selection of exogenous genes or cDNAs in mammalian cell transfections.